Dynamic expansion of mesenchymal stem/stromal cells in a stirred tank bioreactor promotes the release of potent extracellular vesicles

Jan Barekzai; Jonas Friedrich; Maduwuike Okpara; Laura Refflinghaus; Dustin Eckhardt; Peter Czermak; Denise Salzig; Jan Barekzai; Jonas Friedrich; Maduwuike Okpara; Laura Refflinghaus; Dustin Eckhardt; Peter Czermak; Denise Salzig

doi:10.3934/bioeng.2023016

AIMS Bioengineering

2023, Volume 10, Issue 3: 240-264. doi: 10.3934/bioeng.2023016

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Research article

Dynamic expansion of mesenchymal stem/stromal cells in a stirred tank bioreactor promotes the release of potent extracellular vesicles

1.
Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, 35390 Giessen, Germany
2.
Faculty of Biology and Chemistry, Justus-Liebig-University of Giessen, 35390 Giessen, Germany

Received: 13 July 2023 Revised: 18 August 2023 Accepted: 28 August 2023 Published: 06 September 2023

Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are considered a promising therapeutic tool in cell therapy due to their immunomodulatory, regenerative and angiogenic capabilities. However, there is a lack of process knowledge, particularly for a large-scale production of MSC-EV using fully controlled stirred tank bioreactor (STR) systems. For the establishment of a STR-based process, we investigated dynamic process set-ups in spinner flasks, using three different microcarriers, as well as in shaking flasks, using microcarrier-free spheroids. An immortalized cell line (hMSC-TERT) and a particle-free chemically defined medium was used for all approaches. Cell characteristics (e.g., growth, metabolism, cell-specific particle production rates), MSC-EV epitope markers and MSC-EV potency in migration assays were analyzed. We showed that the transfer to a dynamic system (non-porous microcarrier, spinner flask) significantly increased the cell-specific particle production rate (6-fold) and the expression of EV-specific markers. Moreover, MSC proliferation and, most importantly, the therapeutic potency of MSC-derived particles including EVs was maintained. We demonstrated that high cell-specific particle production rates were associated with an increased glucose consumption rate rather than cell growth, which can be utilized for future process development. Furthermore, we showed that dynamic conditions of a controlled 1 L STR significantly increased the cell-specific particle production rate (24-fold) as well as the final concentration (3-fold) of potent MSC-derived particles including EVs. This indicates that fully controlled STRs are an efficient production system for MSC-derived particles including EVs that may open and facilitate the path for clinical applications.
- potent EV production,
- microcarrier-based process,
- large-scale MSC-EV production,
- scratch assay,
- EV analysis
Citation: Jan Barekzai, Jonas Friedrich, Maduwuike Okpara, Laura Refflinghaus, Dustin Eckhardt, Peter Czermak, Denise Salzig. Dynamic expansion of mesenchymal stem/stromal cells in a stirred tank bioreactor promotes the release of potent extracellular vesicles[J]. AIMS Bioengineering, 2023, 10(3): 240-264. doi: 10.3934/bioeng.2023016

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Abstract

Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are considered a promising therapeutic tool in cell therapy due to their immunomodulatory, regenerative and angiogenic capabilities. However, there is a lack of process knowledge, particularly for a large-scale production of MSC-EV using fully controlled stirred tank bioreactor (STR) systems. For the establishment of a STR-based process, we investigated dynamic process set-ups in spinner flasks, using three different microcarriers, as well as in shaking flasks, using microcarrier-free spheroids. An immortalized cell line (hMSC-TERT) and a particle-free chemically defined medium was used for all approaches. Cell characteristics (e.g., growth, metabolism, cell-specific particle production rates), MSC-EV epitope markers and MSC-EV potency in migration assays were analyzed. We showed that the transfer to a dynamic system (non-porous microcarrier, spinner flask) significantly increased the cell-specific particle production rate (6-fold) and the expression of EV-specific markers. Moreover, MSC proliferation and, most importantly, the therapeutic potency of MSC-derived particles including EVs was maintained. We demonstrated that high cell-specific particle production rates were associated with an increased glucose consumption rate rather than cell growth, which can be utilized for future process development. Furthermore, we showed that dynamic conditions of a controlled 1 L STR significantly increased the cell-specific particle production rate (24-fold) as well as the final concentration (3-fold) of potent MSC-derived particles including EVs. This indicates that fully controlled STRs are an efficient production system for MSC-derived particles including EVs that may open and facilitate the path for clinical applications.

Acknowledgments

The authors would like to thank Catharine Meckel-Oschmann for language editing.

Conflict of interest

The authors declare no conflict of interest.

Author Contributions:

J.B. conceived, designed the experiments, and wrote the paper. J.F. wrote parts of the first draft. M.O. and L.R. assisted with the experiments. D.E. helped to draft. D.S. and P.C. revised the manuscript, supervised the research, and acquired funding. All authors have read and agreed to the published version of the manuscript.

References

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